Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.
Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.
复制标题
L-赖氨酸 4-氯酶 BesD 的卤化物和阳离子底物在亚铁和亚铁态下的协同结合。
DOI：
10.1021/acs.biochem.3c00248
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发表时间：
2023
期刊：
影响因子：
2.9
通讯作者：
BollingerJr,JMartin
中科院分区：
文献类型：
--
作者：
Slater,JeffreyW;Lin,Chi-Yun;Neugebauer,MonicaE;McBride,MollyJ;Sil,Debangsu;Nair,MrutyunjayA;Katch,BryceJ;Boal,AmieK;Chang,MichelleCY;Silakov,Alexey;Krebs,Carsten;BollingerJr,JMartin
脂肪族卤代酶需要四种底物：2-氧戊二酸(2OG)、卤化物(Cl-或Br2-)、卤化靶标(“主底物”)和氧气。在经过充分研究的情况下，三种非酸性底物必须结合起来才能激活酶的铁(II)辅助因子，从而有效地捕获O2。卤化物、2OG和(最后)O2都直接与辅因子配位，启动其转化为酰基-卤代-氧代-铁(IV)(卤铁)络合物，该络合物从非配位的基底物中提取氢(H·)，从而实现类自由基碳-卤素偶联。我们剖析了L-赖氨酸-4-氯酶的前三个底物BesD结合的动力学途径和热力学联系。在加入2OG后，卤化物与辅因子的配位以及辅因子附近阳离子赖氨酸的结合都与较强的各向异性协同作用有关。在添加O2时，向卤化铁基中间体的转化不会将底物捕获到活性中心，事实上，卤化物与l-赖氨酸之间的协同作用显著减弱。令人惊讶的是，BesD·[Fe(IV)=O]·Cl·琥珀酸·L-赖氨酸络合物的稳定性使卤代铁基中间体的衰变不会导致1-赖氨酸氯化，特别是在低氯浓度下；一个已知的途径涉及甘油的氧化。这些机制数据表明：(I)BesD可能是在较近的时间或在较弱的选择压力下进化而来的羟基酶祖先，以进行有效的氯化；(Ii)其活性的获得可能涉及在现有羟基酶中失去阴离子蛋白质-羧酸铁配体后，1-赖氨酸结合和氯配位之间的联系。
An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl–or Br–), the halogenation target (“prime substrate”), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme’s Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2all coordinate directly to the cofactor to initiate its conversion to acis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon–halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of thel-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationicl-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O2does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide andl-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result inl-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage betweenl-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.